**Abstract**

Quorum sensing (QS) is a complex system of communication used by bacteria, including several notable pathogens that pose a significant threat to public health. The central role of QS in biofilm activity has been demonstrated extensively. The small extracellular signaling molecules, known as autoinducers, that are released during this process of cell-to-cell communication play a key part in gene regulation. QS is involved in such diverse intracellular operations as modulation of cellular function, genetic material transfer, and metabolite synthesis. There are three main types of QS in bacteria, metabolites of which may form the target for novel treatment approaches. The autoinducing peptide system exists only in Gram-positive bacteria, being replaced in Gram-negative species by the acyl-homoserine lactone system, whereas the autoinducer-2 system occurs in both.

**Keywords:** bacterium, gram-positive, gram-negative, biofilm, quorum sensing, quorum quenching, autoinducer, accessory gene regulator, acyl-homoserine lactone, LuxS, luminescence, *Staphylococcus aureus*, *Vibrio fischeri*, *Vibrio harveyi*

### **1. Introduction**

More than half a century ago, pioneering experiments performed on *Streptococcus pneumoniae* discovered the existence of bacterial communication through hormonelike molecules that were later defined as peptides [1, 2]. These findings were accomplished by studying *Vibrio fischeri*, which is able to produce luminescence at high levels of cell density. The luminous system in this marine bacterium is characteristically self-regulated and is provoked at a threshold level of signal molecules. This so-called "autoinduction" provides an environmental sensing mechanism [3]. Known as "quorum sensing," this type of regulation is a form of information sharing used by bacteria through intercellular communication to regulate gene expression. This process is described in many Gram-positive and Gram-negative bacteria. It is facilitated via autoinducers (AIs) or extracellular signaling molecules that produce, release, and detect as well as respond to them [4]. By increasing bacterial cell density, accumulated AIs in the outer cell will lead to changes in gene expression. This communication is detected in inner species and also between species. Among multiple cell activities that are under the control of QS, biofilm formation, virulence factor formation, sporulation, motility, conjugation, symbiosis, competence, and sporulation are each of note [5–8]. In addition, a number of studies have shown the key role of quorum sensing in metabolic processes, involving a high portion of the bacterial genome (corresponding to more than 20% of the proteome) that facilitates adaptation to metabolic needs [9].

The bacteria belonging to the same colony may exhibit heterogenous phenotypic behavior in order to respond to environmental fluctuations and interbacterial interactions. These are coordinated with each other via quorum sensing, which adapts bacterial traits and behaviors (both group and individual) to ensure their compatibility [10]. In short, QS plays a fundamental role in production, detection, and response to AIs [11].

In the QS system used by various bacteria, there are differences in terms of target genes, types of chemical signal molecules, and mechanisms [8]. Emerging evidence points to several types of signaling molecules, including methyl dodecanoic acid, N-acyl homoserine lactones (AHLs), furanosyl borate, oligopeptides, and hydroxy palmitic acid methyl ester [12]. Although there are multiple QS systems described in bacteria, these are broadly categorized into three groups that we will describe in detail in this chapter. The first major group belongs to Gram-negative bacteria and uses AHLs as the signaling molecule [6]. The second group, only found in Gram-positive bacteria, utilizes small, processed oligopeptides [8]. The third group, in which autoinducer-2 (AI-2) is produced, applies to both Gram-positive and Gram-negative bacteria and has been reported in over 55 species [13].

#### **1.1 Quorum sensing in Gram-negative bacteria**

Some characteristics of QS are common to Gram-negative bacteria. The main feature is the ability of AHLs and s-adenosylmethionine-synthetized molecules to diffuse within the bacterial membrane. The receptors for these are located either in the cytoplasm or on the inner membrane. Additionally, numerous cell processes are affected by QS, which directly modifies the relevant genes [14, 15]. Different types of autoinducers are used by Gram-negative bacteria, whereas the most common type, Acyl-HSL, is found in many bacterial species [14, 16, 17]. The AHLs (lux operon) were first described in *Vibrio fischeri*, which will be discussed as a model in this section [18]. An important reason why *V. fischeri* QS is suitable to study is its high sensitivity to AIs, which means it is activated even when they are at low levels [19–21].

In general, AHL-mediated QS involves either LuxI or LuxR proteins [22]. These are engaged in multiple cell functions including biofilm formation, pathogenesis, antibiotic production, and genetic competence. Hence, LuxI-LuxR is considered an excellent research model [23]. Indeed, the operon LuuxICDABEG is activated by LuxR [22]. More than 20 LuxR analogous families exist in Gram-negative bacteria, of which LuxR is the most studied [24]. LasI and EsaI in *Pseudomonas aeruginosa* and *Pantoea stewartii*, respectively, are of note [25, 26].

LuxR should first be activated by the AIs, N-(3-oxohexanoyl)-L-homoserine lactone (abbreviated to 3-oxo-C6-HSL). This is a diffusible signal catalyzed by a 193-amino acid protein that is encoded by LuxI from a precursor of host metabolism (s-adenosyl methionine) as well as a cofactor acyl carrier protein. In addition to 3-oxo-C6, the other products of LuxI, are apo-ACP and 5′-methylthioadenosine [8, 22, 24, 27, 28]. Thus, in the presence of AI (3-oxo-C6), LuxR activates LuxICDABEG operon expression, and overexpression of LuxR will be followed too [29]. The C-terminal region of LuxR is responsible for DNA-binding as well as RNA polymerase interaction (resulting in activation of the Lux promotor), whereas the N-terminal binds to AIs [30–32].

Other parts of the Lux operon are associated with diverse activities. LuxAB is in charge of encoding luciferase (a heterodimer of two subunits, alpha and beta). LuxC, LuxD, and LuxE are responsible for encoding aldehyde substrate, whereas LuxG

#### *Quorum Sensing in Biofilm DOI: http://dx.doi.org/10.5772/intechopen.113338*

regenerates FMNH2 from FMN [24, 33, 34]. In this regard, luciferase and flavindependent monooxygenase, which produce light photons from chemical energy via catalyzing a bioluminescent reaction, facilitate an enzymatic reaction to produce aliphatic acid (RCOOH) as well as FMN from substrates including FMNH2, O2, and long-chain fatty acids (RCHO). In this way, bacteria regulate luminescence production in light organs of fish at high cell density and switch on *lux* genes (**Figure 1**) [34–37].

Lastly, an intergenic region known as Lux box (a 20-bp palindromic sequence) is located inside the LuxI promoter within 42.5 bp of the LuxICDABEG operon start site. This acts as a transcriptional activator that is responsible for the overexpression of the LuxI promotor [38–40]. Although the Lux box plays an essential part in luminescence gene activation, its precise role and structure remain to be identified [39].

#### **1.2 Quorum sensing in Gram-positive bacteria**

Autoinduction by Gram-positive bacteria is achieved via autoinducer peptides (AIPs) that require postproduction processing. AIPs are not permeable and require carriage across the host cell membrane by transporter proteins [41–43]. Additionally, two types of transcription factors are recognized, Rgg and RNPP, the latter of which is found in all Gram-positive bacteria and is equipped with a binding domain that facilitates its binding to signaling peptides [44].

In the model bacterium *Staphylococcus aureus* (**Figure 2**), there are four types of two-component regulator system, namely, *agr*AC, *sae*RS, *arl*RS, and *srr*AB. Of these,

#### **Figure 1.**

*Lux quorum sensing system in Vibrio fischeri. Autoinducers (AIs) are synthetized by LuxI which later attaches to the LuxR protein at threshold concentration. LuxR protein acts as receptor and its complex with AIs raises LuxI gene expression and thus AI production. AIs diffuse through the cell membrane and thereby activate LuxR protein. LuxCDABEG encodes the structural components of light production in which LuxAB encodes luciferase. Bioluminescence and light production happen after oxidation of RCHO and FMNH2. Production of fatty acids and activation of fatty acyl groups are functions performed by LuxD and LuxC, respectively. Activated fatty acyl groups are then reduced to long-chain aldehydes by LuxE. Recycling of components such as fatty acids as well as FMN, which is produced as a result of the luciferase reaction, are carried out by LuxC/E and LuxG, respectively.*

#### **Figure 2.**

*agr quorum sensing system in Staphylococcus aureus. Autoinducer peptides (AIPs) are produced from the agrD precursor by agrB. Mature AIPs are then exported outside the cell till their concentration reaches a threshold when the two-component system (agrC/agrA) becomes activated. Afterwards, agrA is phosphorylated, enabling it to activate transcription of the P2 and P3 promotors (upregulation). Also, agrA is involved in encoding phenolsoluble modulin peptides (via increasing transcription of psmα and psmβ operons). RNAIII is responsible for regulation of most agr targets as well as delta-toxin (hld).*

accessory gene regulator (*agr*) and *sae* are capable of sensing environmental stimuli, whereas *arl*RS is thought to play a part in antibiotic resistance as well as autolytic activity. The last two-component system, *srr*AB, has a role in energy metabolism and RNAIII inhibition [45]. In addition, *agr* is responsible for controlling virulence factor gene expression by *S. aureus*. The *agr* locus is a density-dependent system, composed of two QS components [46]. There are four main subgroups of *agr*, each of which produces a distinctive AIP. Meanwhile, a two-component system comprising *agr*A and *agr*C is responsible for AIP identification. These AIPs are similar in terms of thiolactone ring structure but differ in amino acid sequence. Moreover, QS is regulated via the *agr* locus, which comprises two transcripts, RNAII and RNAIII. Their expression is induced via P2 and P3 promotors, respectively [45, 47–51]. Activation of *agr* is not limited to AIPs, as additional proteins such as SarA, SrrAB, and other environmental factors can also activate the system [52, 53]. Initially, when *S. aureus* population density is not sufficiently high to induce *agr* expression, colonization occurs through the production of surface proteins, followed by *agr* expression upon increasing cell density. Therefore, *agr* timing adaptation is an indicative of infection progression [54, 55].

In the *agr* system, RNAIII has an important function as an intercellular effector in controlling target gene expression. It also controls other virulence factors, including protein A, Rot protein, leukocidins, enterotoxins, and alpha toxin [50]. Moreover, the four genes *agr*B, *agr*C, *agr*D, and *agr*A are located in the RNAII operon. Initially, *agr*D encodes a 46-amino acid peptide (pro-AIP), which is later processed to yield a 9-amino acid residue. This AIP precursor undergoes modification to C-terminal cleavage before exportation from the cell via *agr*B (a transmembrane endopeptidase). AIP signaling molecules are released into the extracellular environment and have

accumulated there until a threshold concentration is reached when they are detected by specific sensors. *agr*C, a transmembrane histidine kinase protein, is phosphorylated and attaches to AIPs, thereby enabling gene regulation in QS to be followed. This is also responsible for the activation of *agr*A, which is a response regulator [8, 49, 50, 56, 57]. In an autofeedback cycle, upregulation of RNAII and RNAIII transcription is driven by the binding of *agr*A to P2 and P3 promoters, respectively [50]. In short, the simultaneous activation of *agr*A and *agr*C, which act as transcription factors for RNAIII, induces the RNAII operon [49, 58]. Upon activation, RNAIII can trigger the production of alpha toxin. Meanwhile, the RNAIII is able to quench the expression of certain surface virulence factors (including coagulase, peptide A and FNPA, B; [59].

A further activation pathway has been reported in various Gram-positive bacteria. This involves interaction between signaling molecules and receptors inside the cell, after which the expressed products are transported to the external environment [60, 61]. This is exemplified by *Enterococcus faecalis*, in which the interaction between peptides and PrgX proteins alters the activity of conjugative plasmids [62, 63] and by Phr peptides acting as phosphatase inhibitors in *Bacillus* species [62, 63]. Finally, a strong relationship between *agr* and σ<sup>B</sup> , a biofilm formation regulator, has been identified [64]. Formation and dispersal of biofilm are associated with the downregulation and upregulation of *agr*, respectively [65, 66].

#### **1.3 Autoinducer-2 in Gram-positive and Gram-negative bacteria**

AI-2 is found in both Gram-positive and Gram-negative bacteria, where it facilitates intra- and inter-species communication [67, 68]. AI-2 signals have been described as providing an "interconversion nature", meaning that this molecule is utilized by different bacteria as a universal tool for communication [68]. Support for this notion comes from the observation that, unlike for single-species oral biofilm formation, in mixed populations of *Porphyromonas gingivalis* and *Streptococcus gordonii*, LuxS expression by each species is required. Further evidence shows that if there is a deficiency of *Streptococcus mutans,* other species of oral bacteria supplement with *luxS* mutation in biofilm formation [69].

In this system, the enzyme LuxS catalyzes the synthesis of AI-2 or its precursor 4,5-dihydroxy-2,3-pentanedione [70]. Two receptors, LuxP (a periplasmic-binding protein) and LsrB, are detected. Biofilm formation, virulence factor production, and other density-dependent phenotypes are attributed to the former, with delivery of AI-2 into cells ascribed to the latter [67, 70, 71]. They differ in structure, exemplified by LuxP-AI-2 in *Vibrio harveyi* being composed of furanosyl borate diester, whereas LsrB-AI-2 in *Salmonella typhimurium* lacks boron [71, 72]. Molecular analysis indicates that the type of AI-2 varies with bacterial species [72]. Multiple bacteria have been identified that can react to AI-2, including *Staphylococcus epidermidis*, *Helicobacter pylori*, *Bacillus subtilis, Pseudomonas aeruginosa*, *Campylobacter jejuni*, *S. mutans,* and *Listeria monocytogenes* [73–79]. To date, most information on this system comes from *V. harveyi* [69], for which three-channel quorum sensing is proposed, involving AI-1, AI-2 and, cholerae AI-1 [80].

The *V. harveyi* protein LuxQ has a cytoplasmic histidine-kinase domain, a response regulatory domain, and a periplasmic sensor domain. Interestingly, upon binding to the AI-2, LuxQ functions as a kinase and as a phosphatase at low and high cell densities, respectively [70, 81]. Another protein known as LuxP is able to modify LuxQ activity (through a histidine-kinase sensor), and it is this union that regulates

#### **Figure 3.**

*AI-2 quorum sensing system in Vibrio harveyi. The three autoinducers HAI-1, AI-2 and CAI-1 are synthesized by LuxM, LuxS and CqsA, respectively. LuxS produces AI-2 by converting S-ribosylhomocysteine (SRH) to dihydroxypentane-2,3-dione (DPD) in the cell cytoplasm. This occurs when LuxS participates in the activated methyl cycle, which generates and recycles methyl donors. DPD is a by-product of the LuxS reaction that produces SRH. Later, DPD undergoes cyclization and rearranges without enzymatic support to produce AI-2 prior to export across the outer membrane. A two-component signal regulator is responsible for the responding pathway in vibrio spp., while for Salmonella enterica this is identified as an ABC transporter. In V. harveyi, furanosylborat-diester and periplasmic LuxP together form active AI-2, inducing phosphatase activity in LuxQ. This leads to phosphate transfer from LuxU to LuxO, which is the response regulator. Finally, several cell changes take place, including bioluminescence. In contrast, when cell density is low and there is no AI-2, phosphorylated LuxO as well as σ54 produce small regulatory RNAs. Their interaction with LuxRVh mRNA causes destabilization of Hfq-dependent chaperone proteins. This results in suppression of transcription of the lux operon and a reduction in bioluminescence. Meanwhile, dephosphorylated LuxO, the level of which increases in the presence of AI-2, reverses the flow of phosphate.*

the AI-2 QS regulon (**Figure 3**) [70, 82]. Following the conversion of LuxQ activity from kinase to phosphatase via the transmembrane sensor histidine kinase, LuxP bound to AI-2 regulates gene expression of phenotypes such as biofilm formation and bioluminescence [67, 70, 83].

### **2. QS and biofilm**

Multiple factors benefit bacterial colonies that adopt a multicellular lifestyle rather than remain planktonic. Bacterial cells embedded within biofilm are protected from detrimental factors, whereas nutrient-deficient conditions and hostile environments are both noted among driver factors for biofilm production [84]. A crucial component of mature *S. aureus* biofilm is the extracellular matrix. This is composed of eDNA, polysaccharide intercellular adhesin, and other proteins. It is the most stable, thus, a problematic stage to treat. To reduce biofilm mass, detachment follows, in which QS as well as nuclease and protease enzymes play a significant part [85–88]. The cell-tocell signaling of QS pertains to all biofilm formation stages. A key role in the initiation is the communication between bacteria through the detection of AIPs [89]. Chronic

infection of *S. aureus* as well as biofilm formation is linked with low activity of *agr* QS [50]. *In vivo* studies have demonstrated the importance of the *agr* system to disease progression. Although upregulation by *agr* has a role in acute infections, downregulation is involved in biofilm formation [66, 90, 91]. In the dispersal stage, which is directly under-regulation of *agr*, isolation of new cells is ascribed to P3 promoters via the production of proteases and glucose depletion [92]. Hence, there is a direct relation between QS activation and the transition between biofilm and planktonic cell lifestyles. Thus, it remains to be determined whether QS quenching results in biofilm blockage [93, 94].
